(CANCER RESEARCH 48, 3292-3296, June 1, 1988]

Expression of a-Amylase in Human Lung CancersNaohiro Tornita,1 Nariaki Matsuura, Akira Horii, Mitsuru Emi, Takahiro Nishide, Michio Ogawa, Takesada Mori,

Osamu Doi, and Kenichi Matsubara

Institute for Molecular and Cellular Biology, Osaka University, Vainada oka, Suita, 565 [N. T., A. H., M. E., T. N., K. MJ; Second Department of Surgery, OsakaUniversity Medical School, Fukushima-ku, Osaka 553 [N. T.,N.M.,A.H.,M. E., T. N., M. O., T. M.J; and Department of Surgery, The Center of Adult Diseases, Osaka,Higashinari-ku, Osaka 573 [O. D.J; Japan

ABSTRACT

Thirty-three human lung tumors were studied for the expression of «-amylase by immunohistochemical and Northern blot analyses. Twenty ofthem were adenocarcinomas, among which 17 proved to be adequate formRNA analyses and were, except for two, amylase mRNA producers.Seven were squamous cell carcinomas, none of which producedamylase.The remainingsix consisted of two undifferentiatedsmall cell carcinomas,and one each of undifferentiated large cell carcinoma, carcinoid tumor,mucoepidermoid carcinoma, and metastatic lung cancer; the mucoepider-moid carcinoma proved to be an amylase producer. These observationsstrongly suggest that among lung cancers, the production of «-amylaseis a propertycommonly associated with adenocarcinomasand can be usedfor distinguishing cell types. Histogenesis and carcinogenesis in lung cellsare discussed in connection with the cells that produce amylase.

INTRODUCTION

Human a-amylase, which hydrolyzes a-l,4-glucosidic bonds,is mainly produced in the salivary gland and pancreas. Amylaseis also released into serum and urine when the patient carries aspecial type of malignant neoplasm, such as lung cancer, ovarian cancer, or thymoma, even when the patient has no disorderof the salivary gland or pancreas. Since Weiss et al. (1) firstreported elevated serum amylase levels caused by bronchogeniccarcinoma in 1951, several articles have appeared reportingsimilar cases of hyperamylasemia in patients with primary lungcancer (2-19). This amylase, studied by biochemical means,was shown to be mostly the salivary type isoamylase (5-12, 14-19). Electron microscopic studies with these tumor cells showednumerous electron dense bodies, which are typical for secretorygranules (5, 7, 9, 15).

The ectopie expression of amylase gene in malignant cellsaroused much interest, but the mechanism of "gene activation"

is not yet understood, since knowledge about the genes foramylase production is limited. Recently, we cloned amylasecDNA2 from a lung adenocarcinoma and showed that theamylase mRNA was of the salivary type.3

Histologically, most lung cancers which produce amylasehave been shown to be adenocarcinomas, but there are reportswhich claim that squamous cell carcinomas (18) and undifferentiated carcinomas (5, 14, 19) may also produce hyperamylasemia, although these studies did not provide direct evidence toshow that the cancer cells themselves produce amylase.

Hyperamylasemia in patients with primary lung cancer hasbeen reported in three out of 33 cases (2) and in six out of 55cases (16). However, the exact frequency of lung cancers whichexpress amylase has not been precisely established.

Received 7/8/87; revised 11/13/87. 2/19/88; accepted 2/26/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom all correspondence and reprint requests should be addressed, atInstitute for Molecular and Cellular Biology, Osaka University 1-3, Yamada-oka,Suita, Osaka 565, Japan.

2The abbreviations used are: cDNA, DNA complementary to mRNA; PAP,peroxidase antiperoxidase; SSC, standard saline citrate (IxSSC = 0.15 M NaCI/IS HIMNa citrate); SDS, sodium dodecyl sulfate.

3Nakamura et al,, manuscript in preparation.

Based on these findings, we initiated systematic studies withamylase gene expression in lung cancers with particular emphasis on solving the following problems: (a) What fraction of lungcancers express amylase? (¿>)Is there any correlation betweenthe histological type of lung cancer and the ability to expressamylase? (c) Are amylases expressed in lung cancers all of thesalivary type?

Our results show that among lung cancer cells, amylasemRNA was produced at various levels in almost all the adenocarcinomas, which constituted more than half of the resectedlung cancers. Thus, this mRNA synthesis may be taken as amarker for classifying cell types among lung cancers.

MATERIALS AND METHODS

Tissue Samples. Tissue samples of lung tumors were obtained duringsurgery from 33 patients, that included 24 men and 9 women (ages 35-76). The surgical operations were done in four different clinics, butmostly at the Department of Surgery, the Center of Adult Diseases,Osaka. The patients' characteristics and the record of histological

diagnoses are listed in Table 1.A part of the resected tumor tissue (0.5-3.0 g) was immediately

frozen in liquid nitrogen, except for a small aliquot that was assayedfor immediate amylase activity, and another small aliquot that wasfixed in 10% neutral buffered formaldehyde for immunohistochemicalanalysis. The frozen samples were kept at —70°Cuntil use within 1

month for the preparation of mRNA. Nontumorous portions of lungtissues were processed in the same manner and were used as normalcontrols.

Assay of Amylase Activity in Tumor Tissue. This was done by thechromogenic method described by Ceska et al. (20) using blue starchpolymer (Pharmacia Co., Sweden) as substrate. Because of the limitedsupply of tissue samples, a small amount of sample, usually 50 mg, wasused for this test. Activity was expressed in IO"2 Somogyi units/g wet

tissue.Immunohistochemistry. Immunohistochemical study was done using

the PAP method described by Sternberger (21). The rabbit anti humana-amylase antiserum was prepared in our laboratory and its specificityto a-amylase has been described (22). The formaldehyde-fixed, paraffin-embedded tissues were cut 4-um thick, deparaffinized using xylene,réhydratée.)in a graded series of ethanols, and then immersed in meth-anol containing 0.03% hydrogen peroxide for 30 min in order to blockendogenous peroxidase activity. The sections were washed twice in 0.05M phosphate buffered saline, pH 7.2 (15 min each) and incubatedovernight at 4°Cwith the primary antiserum to human a-amylase

(1:800). The sections were then incubated with swine anti-rabbit im-munoglobulins (1:20) for 30 min, after which the PAP complex (1:40)was applied for 30 min at room temperature. Between each incubation,the sections were washed three times (10 min each) in phosphatebuffered saline. The peroxidase reaction was developed by adding 0.02%3-3'-diaminobenzidine, 0.005% hydrogen peroxide in 50 mM Tris-Cl

(pH 7.6) for 3 to 10 min. The reaction mixtures were counterstainedwith hematoxylin or methyl green.

Human pancreas tissue was run in parallel as a positive control andnonimmunized rabbit serum was used as the negative control.

Preparation of Amylase cDNA Probe. A 1.6-kilobase pair Xhol fragment of pAM82 (23) was used as a probe. This DNA covers the entireamino acid coding region of salivary type amylase cDNA (24). A 32P-labeled cDNA probe (specific activity, 4-8 x 10" cpm/ug) was prepared

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EXPRESSION OF «-AMYLASEIN HUMAN LUNG CANCERS

Table 1 Individual data for 33 patients with lung cancer including results of assay for amylase activity, immunohistochemical, and Northern blotting records

CaseAdenocarcinoma1234567891011121314151617181920Squamous

cellcarcinoma21222324252627Undifferentiated

small cellcarcinoma2829Undifferentiated

large cellcarcinoma30Carcinoid31Mucoepidermoid

carcinoma32Metastasis

of renal cellcarcinoma33Age

andsex71

F35M69M59M60M60F74

M73F53

M76F71

F65M73M62M64M52M50F63

M51M65F58

M64M66M58M73M62M50M47F66

M58

M61

F67

M70

MHistology"well

papwellpapmodtubmodpapwelltubwellpapmodpapwellpapwellpapportubmodpapmodtubmodpapwellpapwellpapwellpapporsolidporsolidmodpapmodpapwellwellmodwellpormodwellSerum

amylase*5964NDND75762288477561401121697885748170117168108779575120121111106121507461110Tissueamylase'3902202101600270950802904204602309780100360618060069049011800340100260390200900170100320290360Immunohistochemistry1'Northernblotting'ND''

+ND

+ND++

+++ND++ND+++±-•++±+++—+++++++±-*+++—-f++++++—

———————--————±-

-—

—++

-±

+ND

" well, well differentiated; mod, moderately differentiated; por, poorly differentiated; pap, papillary; tub, tubular. The two small cell carcinomas (Cases 28 and 29)

are of the intermediate cell type.* Serum amylase level is expressed in IU/liter. The normal range is 50-145.c Amylase activity in tumor tissue is expressed in IO"2 Sotnogyi units/g wet tissue.rfThe intensity of staining was graded as follows: ++, strong; +, moderate; ±,weak; -, none. Amylase expression in adenocarcinoma ('Via) versus squamous cell

carcinoma (%) is statistically significant (P < 0.001) by contingency analysis, x22 x 2.' +, the transcript for «amylase was detected; -, not detected even after several days of exposure; *, the mRN A was degraded as shown by rehybridization of the

same filter with "P-labeled 7-actin probe. Amylase expression in adenocarcinoma ("/n) versus squamous cell carcinoma (%) is statistically significant (P < 0.001).'ND, not done.

by the random priming method (25) using [a-32P]dCTP (Amersham,

3000 Ci/mmol; 1 Ci = 37 GBq).Preparation of mRNA. Total cellular RNA was isolated from tissues

essentially as described previously (26). mRNA was purified from thetotal RNA by repeated passages through oligo-dT cellulose (Type 7;

Pharmacia Co., Sweden) column.Northern Blot Analyses. An aliquot (5 ¿ig)of mRNA was denatured

by heating at 65°Cfor 15 min in 50% (vol/vol) formamide, electropho-

resed in a 1% agarose/2.2 M formaldehyde gel as described (27), andthen transferred to a nylon filter (Gene Screen Plus, NEN). After beingbaked at 80"C for 2 h, the filter was prehybridized at 65°Cfor several

hours in a sealed plastic bag containing 6xSSC/l % SDS. Hybridizationwas carried out at 65"C overnight in 6xSSC/l% SDS/heat-denaturedherring sperm DNA (20 /ig/ml) containing amylase cDNA probe (1-2x IO6cpm/ml) described above. After the hybridization, the filter wasrinsed at room temperature in 2xSSC/l% SDS, washed twice at 65°C

in the above solution for 30 min., and then rinsed at room temperaturein O.lxSSC. The filter was kept wet and exposed to X-ray film (KodakXAR-5) overnight at -70"C with intensifying screen. For detecting

very weak signals, exposure was continued for 2 to 7 days.mRNA degradation was monitored using a 7 act in probe. The filters

were washed twice at 90°Cin O.OlxSSC/0.01% SDS to remove the

amylase probe, and then rehybridized with 7-actin, whose transcriptwas about 1.9 kilobase pair in size. Since a 184-base pair Hincll-Xbal

fragment prepared from S12 is highly homologous to 7-actin,4 this

DNA fragment was used as a probe.

RESULTS

Table 1 shows the individual data for 33 patients that weanalyzed. The 33 cases consisted of 20 adenocarcinomas, 7squamous cell carcinomas, 2 Undifferentiated small cell carcinomas, and one each of Undifferentiated large cell carcinoma,carcinoid tumor, mucoepidermoid carcinoma, and metastaticlung tumor from renal cell carcinoma.

Serum Amylase Levels. Serum amylase levels were measuredbefore operation. All the patients had neither pancreatic norsalivary gland diseases. No renal insufficiency was detected.

The data are assembled in Table 1. Fig. 1 shows the scatter-gram of serum amylase levels. Twenty-seven of the 31 casessubjected to measurements showed serum amylase levels withinthe normal range. Three cases (Cases 7, 13, 20) which displayedhyperamylasemia were all adenocarcinomas.

4 M. Emi, A. Morii, N. Tornita, T. Nishide, M. Ogawa, T. Mori, and K.

Matsubara. Overlapping of two genes in human DNA: a salivary amylase geneoverlaps with a r-actin pseudogene that carries an integrated human endogenousretroviral DNA. Submitted for publication.

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MO-IBO-100-A.carcinoma-fB.cellcarcinoma•«

*•C.':

Fig. 1. Scattergram depicting the serum rumiase levels. A, adenocarcinomas.Cases 1, 2, 5-20 (/V = 18); B, squamous cell carcinomas, Cases 21-27 (N = 7);C, other carcinomas, Cases 28-33 (N = 6). Two horizontal lines, normal range.

Fig. 2. Scattergram depicting the amylase activity in normal and tumor tissues.A, nontumorous lung tissues obtained from Cases 4, 16, 17, 19, 20, 26, and 32(N = 7); B, adenocarcinomas, Cases 1-20 (A' = 20); C, squamous cell carcinomas,Cases 21-27 (/V= 7); D, other carcinomas. Cases 28-33 (N = 6).

Amylase Activity in Tumor Tissues. Tissue amylase activitywas assayed with the resected cancers (Fig. 2). Six were foundto have high amylase activity (>500 X 10~2 Somogyi units/g

wet tissue), including the three hyperamylasemia. These sixamylase producing cancers were adenocarcinomas (Table 1).No elevated amylase activity was detected in any of the squamous cell carcinomas and other carcinomas. Because of limitedtissue supply, further assays using larger amounts of tissuescould not be done.

Immunohistochemical Observations. The cancer tissues weresubjected to immunohistochemical examinations using the PAPmethod that employs rabbit anti-human a-amylase (see "Materials and Methods"). The data are assembled in Table 1, and

some of the pictures are given in Fig. 3. Positive granularstaining for amylase can be seen in the apical cytoplasm of allmalignant cells in Case 13 which is a moderately differentiatedpapillary adenocarcinoma (Fig. 3, A and B). No staining foramylase is encountered in Case 22 which is a well-differentiatedsquamous cell carcinoma (Fig. 3C). Weak staining in about20% of the malignant cells can be noted in Case 32 which is amucoepidermoid carcinoma (Fig. 3D).

Altogether, 18 of the 28 lung cancer tissues which had beensubjected to the immunoreactivity against anti a-amylase

B

D

Fig. 3. Immunohistochemical demonstration of amylase in lung cancers. . I.moderately differentiated papillary adenocarcinoma (Case 13). Almost all malignant cells are positive for amylase (xllS.S). B, higher magnification of thespecimen as shown in. I (Case 13). A positive granular staining can be seen in theapical cytoplasm of malignant cells (x700). C, well-differentiated squamous cellcarcinoma (Case 22). No positive staining can be seen (X175). O, mucoepidermoidcarcinoma (Case 32). A few malignant cells show weak staining (X560). All tissueswere stained with anti a-amylase antibody using the PAP method and counter-stained with methyl green.

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showed positive results. Among these, 15 out of the 16 testedwere adenocarcinomas. In addition, one carcinoid tumorshowed intense staining and one each of the undifferentiatedsmall cell carcinomas and the mucoepidermoid carcinomashowed weak staining. On the other hand, all seven squamouscell carcinomas gave negative results. One undifferentiatedsmall cell and a large cell carcinoma showed no staining. Thefraction of cells that were stained and their staining intensityvaried depending upon the tumor tissue, although their stainingpattern, particularly the diffuse granular staining pattern incytoplasms, was almost the same.

The intensity of staining had no correlation with the extentof differentiation of the adenocarcinoma.

Northern Blot Analyses. The results with 14 samples areshown in Fig. 4. Five ng of mRNA from tumor tissues weretested, except in Case 32 where 10 ¿igwas used. The controllanes a and b in Fig. 4 were loaded with 0.02 /¿gmRNA fromthe normal salivary gland and the pancreas, respectively. Thesalivary type mRNA is 1.8 kilobase pairs in size, whereas thepancreas type amylase mRNA is 1.6 kilobase pairs in size (24).

Among the samples tested, all the adenocarcinomas (Cases1, 4, 7, 13, 15 and 17) had a 1.8-kilobase pair transcript thatreacted with the amylase cDNA probe, the size coinciding withthe salivary type amylase mRNA. On the other hand, no hybridizing bands were found for the squamous cell carcinomas(Cases 22,23, and 24), the undifferentiated small cell carcinoma(Case 28), the undifferentiated large cell carcinoma (Case 30),the carcinoid tumor (Case 31), or the metastatic carcinoma(Case 33). Fig. 4, lane c, containing 5 ¿igmRNA from anoncancerous part of the lung, showed no detectable band. Themucoepidermoid carcinoma (Case 32) displayed a hybridizingband of the same size as those in the adenocarcinomas. Similartests were done with the other samples, except for Case 5. Theresults are listed in Table 1. Altogether, 15 of the 19 adenocarcinomas examined showed transcripts of amylase. Two of thefour cases which showed no hybridizing band were shown tohave degraded mRNA, as revealed by rehybridization of thesame filter with -y-actin probe. Omitting these two samples, ourresults show that 15 of the 17 adenocarcinomas expressedamylase. Only two cases (Cases 3 and 12) showed no amylasemRNA, although, the Case 12 sample showed a positive im-munohistochemical sign, this discrepancy can not be explainedat present. Judging from the density of the hybridizing bands,there was great variation in the amounts of amylase mRNA inthese adenocarcinomas. The relative level of amylase mRNAwas estimated to be about 1/200 ~ 1/2000 of that in the

pancreas.In contrast to these adenocarcinomas, none of the seven

squamous cell carcinomas showed any sign of amylase expression.

DISCUSSION

This is, to our knowledge, the first report of an extensivestudy on the expression of a-amylase in various lung cancers.The results clearly show that practically all adenocarcinomasproduce amylase, although the extent of expression varies withthe tissue. In contrast to the adenocarcinomas, none of thesquamous cell carcinomas showed any sign of amylase expression. Lung cancers are markedly heterogenous in morphologyand in biochemical character. Some of them produce substanceslike peptide hormones, enzymes, or mucin. Due to the multidirectional and heterogenous cell differentiation in lung cancers, production of these substances has not been classified into

histológica! types. For example, the secretory component ofIgA which is produced from exocrine cells (28) can be demonstrated in many lung adenocarcinomas, but it is also detectedin some squamous cell carcinomas or undifferentiated carcinomas. Our data raise the possibility that a-amylase might serveas a marker for adenocarcinomas among lung cancers. In onecase of metastatic tumor from renal cell carcinoma, amylaseexpression was not shown. To our regret, we could not obtainlarger numbers of samples of metastatic lung cancer. We studiedfor amylase expression in 13 cases of primary adenocarcinomasof stomach, colon, pancreatic duct, gallbladder, and liver byimmunohistochemical analysis, and none of them showed amylase expression (data not shown). So the prediction is thatmetastatic adenocarcinomas in lung would not express amylase.With further support from analyses of larger numbers of samples, a-amylase expression might serve as a useful marker inclinical work, for instance in differentiating between primarylung adenocarcinomas and metastatic adenocarcinomas.

One case of mucoepidermoid carcinoma, which is rare amonglung cancers, showed expression of amylase. This is an interesting result because mucoepidermoid carcinomas are considered to possess characteristics of both adenocarcinoma andsquamous cell carcinoma.

Amylase was detected in a sample of undifferentiated smallcell carcinoma and carcinoid tumor by immunohistochemicalmeans. This might be regarded as evidence that such cancercells have the potential of multidirectional cell differentiation.

There are some discrepancies in the detection of amylase byimmunohistochemical means and Northern blottings, as shownin Cases 12, 18, 28, and 31. These discrepancies may reflectthe difference in detection threshold of the two methods, or theheterogeneity of tissue samples, as demonstrated by unevendistribution of staining cells in immunohistochemistry. Eventhough it is not likely, the possibility can not be ruled out thatcancer cells not producing amylase may take it up and store it.Further work is needed to clarify these points.

Recently, we cloned and sequenced the amylase cDNA froma lung adenocarcinoma, and showed that it was of the salivarytype.3 In the present study, Northern blotting results showed

that the size of the amylase mRNA of lung cancers was 1.8kilobase pairs, coinciding with that of salivary type mRNA.These observations indicate, in accordance with previous biochemical studies of amylase in tumor tissue (5-12,14-19), thatamylase in lung cancer is, in almost all cases, the salivary type.

In some lung cancers, especially in small cell lung cancers,gene amplification and elevated expression of c-myc (29) or N-myc (30) oncogene have been reported. An intriguing possibilityis that amylase expression in lung cancer may reflect amylasegene activation in association with carcinogenesis. Southernblot analysis of DNA obtained from the Case 13 sample showedthat the amylase gene(s) had undergone neither amplificationnor rearrangement (data not shown).

Recently Hayashi et al. demonstrated by histochemical andimmunohistochemical methods that ciliated epithelial cells andserous glandular cells of the bronchus, not the alveolar epithelium, seem to produce amylase (31). We tried to detect amylasemRNA using as much as 40 fig of normal lung mRNA inNorthern blotting, but no positive signal was detected (data notshown). However, these discrepancies can be reconciled byassuming that the lung carries a very small fraction of amylase-producing cells. If we accept this idea, the expression of amylasegene in the lung may not be "ectopie" but must be reevaluated

in the light of histogenesis and carcinogenesis of the lungcancers. There is a good possibility that lung adenocarcinomas

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EXPRESSION OF «-AMYLASE IN HUMAN LUNG CANCERS

Fig. 4. Northern blot profiles of mRNAsisolated from various lung cancer tissues. Thesample No. is given above each lane. For de- Case No.tails, see Table 1. Cases 1, 4, 7, 13, 15, 17,adenocarcinomas; Cases 22, 23, 24, squamouscell carcinomas; Case 28, undifferentiatedsmall cell carcinoma; Case 30, undifferentiatedlarge cell carcinoma; Case 31, carcinoid tumor;Case 32, mucoepidermoid carcinoma; Case 33,metastatic carcinoma. A portion (5 fig) ofmRNA, except in Case 32 where 10 ng wasused, was electrophoresed in a 1% agarose gelcontaining 2.2 M formaldehyde, transferred toa nylon filter, and hybridized with 32P-labeled 1.8kbamylase cDNA probe as described in "Materials and Methods." The controls in lanes a, b, 1.6kb

and c carried, respectively, salivary glandmRNA (0.02 ,ig), pancreas mRNA (0.02 ¿ig),noncancerous lung mRNA (5 >ig). Exposurewas done at —70"Cwith an intensifying screen

for 14.5 h (control samples a, b, and c. Cases13, 31, 33), 26 h (Case 7), 45 h (Case 32), 4days (Cases 1, 4,17, 22, 23, 24), 1 days (CasesIS, 28, 30). kb, kilobase pair.

1 4 7 13 15 17 22 23 24 28 30 31 32 33

originate from cells which are normally or potentially capableof producing amylase. Expression of a-amylase in lung cancercells may reflect the differentiation tendency of these cancercells to exocrine cells of the bronchial epithelium, althoughthere is no direct evidence for this. Further studies on morelung cancers, as well as on normal lung tissue cells are needed.

ACKNOWLEDGMENTS

We thank Drs. K. Kodama and M. Tatsuta at the Center of AdultDiseases, Osaka, E. Kurokawa at Minoo City Hospital, K. Kagotaniand S. Noguchi at Nishinomiya Municipal Central Hospital, T. Kanekoand K. Okagawa at Kinki Central Hospital of the Mutual Aid Association of Public School Teachers, for supplying the samples. We alsothank Dr. Y. Takahashi for his technical advice and Miss M. Inoue forher secretarial assistance.

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